PB2071: SENSITIVITY OF MULTIPLE MYELOMA TO MELFLUFEN ASSOCIATES WITH DECREASED P53 ACTIVITY AND ENRICHMENT OF DNA DAMAGE REPAIR PATHWAY GENES
Authors: Juho Miettinen, Philipp Sergeev, Sadiksha Adhikari, Maiju-Emilia Huppunen, Minna Suvela, Ana Slipicevic, Nina N Nupponen, Klara Acs, Fredrik Lehmann, Caroline Heckman
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: The novel peptide-drug conjugate melphalan flufenamide (melflufen) was recently approved by the EMA for the treatment of relapsed/refractory multiple myeloma (MM). Considering the low overall survival of these patients and the challenges to decide their treatment, information that can support treatment selection and predict outcome is urgently needed. Aims: We aimed to identify potential indicators of melflufen response in MM as well as mechanisms underlying the sensitivity by applying ex vivo multiparametric flow cytometry-based drug sensitivity and resistance testing (DSRT) and single cell RNA sequencing (scRNAseq) to MM patient samples. Methods: Bone marrow aspirates were collected and mononuclear cell fractions (BM-MNCs) prepared from 12 newly diagnosed (ND) and 12 relapsed/refractory (RR) MM patients after written informed consent following approved protocols in compliance with the Declaration of Helsinki. For DSRT the MM BM-MNCs were seeded onto 96-well plates and treated with 7 different concentrations of melflufen or melphalan. After 72 h incubation the cells were stained for CD138 and CD38 to identify plasma cells and Annexin V and 7AAD to distinguish apoptotic and dead cells. For scRNAseq, BM-MNCs were sorted based on CD138 expression and the CD138+ cells mixed with CD138- cells at a 1:1 ratio. Sequencing libraries were prepared using 10x Genomics reagents and instrument followed by sequencing on the Illumina NovaSeq 6000. The samples were grouped based on sensitivity to melflufen and melphalan: high sensitivity (HS, DSS > 40 (melflufen) or DSS > 16 (melphalan)), intermediate sensitivity (IS, 31 ≤ DSS ≤ 40 (melflufen) or 10 ≤ DSS ≤ 16 (melphalan)), and low sensitivity (LS, DSS < 31 (melflufen) or DSS < 10 (melphalan)). Differential gene expression (DGE) and gene set enrichment analysis (GSEA) between the HS and LS samples for both drugs were performed to identify genes and pathways associated with drug response. Results: We analyzed the DSRT, scRNAseq, and associated clinical and cytogenetic data for the MM samples of different melflufen sensitivity groups (HS, IS, LS). A significant indicator of melflufen sensitivity was disease stage as all but one of the HS samples were from RRMM patients. All HS samples with cytogenetics had a gain of 1q, while all LS samples had a deletion of 13q. In addition, 4 of the 8 LS samples and only 1 of the 8 HS samples had t(4;14), while 3 of the HS and 2 of the LS samples were positive for del17p. Mutation to TP53 was detected in 2 HS samples suggesting that melflufen is active despite loss of p53 activity. In support of this, GSEA of the DGE results from plasma cells of the HS samples showed a decrease in p53 downstream pathway and targets. In contrast, the same analyses showed higher enrichment of pathways associated with DNA damage repair genes BRCA1, ATM, and CHEK2. GSEA of the DGE results from healthy cells detected in the HS samples revealed differential enrichment of innate immune system and interferon signaling pathways. Summary/Conclusion: We demonstrate that meflufen is highly effective at inducing cell death of plasma cells ex vivo, particularly in samples from RRMM patients. Importantly, melflufen was active in samples from patients with high-risk features such as del17p, 1q gain and TP53 mutation. Associated with this was a decrease in the p53 pathway and increase in other DNA damage repair genes. Keywords: Gene expression, Drug sensitivity, Multiple myeloma